In muscle and nonmuscle cells the molecular motor myosin plays crucial roles in contraction, various forms of cell movement, and changes in cell shape. In spite of decades of investigations on myosin, the molecular basis of how the chemical energy of ATP hydrolysis is converted into mechanical movement is not understood. The development of quantitative in vitro assays for myosin movement along actin filaments has provided a new powerful tool to examine the molecular basis of this movement. Of these assays, the 'myosin-coated surface assay' has proved the most useful. These developments were the foundation of the origin of this grant. The specific aims of this competing renewal are to develop more refined in vitro assays for myosin movement and to use these assays and the actin- activated myosin ATPase assay to define the mechanical and biochemical parameters of native and mutated forms of myosin. We propose to develop the in vitro motility assay to measure individual steps in the motion produced by one or a few myosin molecules. We also propose to develop an assay for measuring force produced in vitro using purified actin and myosin. We have already used molecular genetic approaches to express large amounts of functional forms of native Dictyostelium myosin and its head fragments in Dictyostelium. Mutated forms of myosin and its fragments will be obtained using the same approaches. These various forms of myosin and its head fragments will be purified to homogeneity and will then be analyzed in vitro using actin-activated ATPase assays and in vitro motility assays to determine parameters of both velocity and force. Such correlations of ATPase activity, force, and velocity should lead to a better understanding of the molecular basis of myosin movement.